Biomedical Engineering Reference
In-Depth Information
sodium phosphate buffer for pH 7.0 and 7.4). The buffering agents were prepared as
concentrated stock solutions. Then, appropriate volumes of them were mixed with the
samples of cell-free medium to obtain a final buffer concentration of 0.1 M. The samples
containing the amylase were incubated for 5 min at the corresponding temperature according
to the experimental matrix defined by the design used (Table 1). The enzyme activities
obtained were corrected with the corresponding dilution factor.
Results were analyzed by Experimental Design Module of the Statistica software package
(Statistica 5.1 for Windows computer program manual; StatSoft Inc. Tulsa, OK, USA). The
response surfaces were plotted using the DeltaGraph software, version 4.0 (SPSS, Inc.,
Chicago, IL, USA).
Solid State Fermentation (SSF)
Sugar cane bagasse was treated with NaOH (0.12 g of NaOH per gram of dry bagasse)
and autoclaved at 121ºC for 20 min (Gutiérrez-Correa & Tengerdy, 1997) to remove the core
and noncore lignin fractions (Doran et al., 1994). Then, the samples of sugar cane bagasse
were thoroughly washed with tap water, subsequently with distilled water until neutrality and
dried at 80ºC (Gutiérrez-Correa & Tengerdy, 1997).
Fermentations were conducted in 250-mL Erlenmeyer flasks containing 5 g of dried
bagasse supplemented with the basal salt solution, previously adjusted at the appropriated pH
value, and distilled water until reaching the desired moisture level. The salt solution contained
(%, g/g of dry support), NH 4 NO 3 , 1; KH 2 PO 4 , 1; NaCl, 0.2; MgSO 4 7H 2 O, 0.2 (Francis et al.,
2003). The contents of the flasks were thoroughly mixed and autoclaved at 121ºC for 15 min.
After cooling, the flasks were inoculated with 1.25 mL of inoculum containing the
appropriate cell suspension. The contents were thoroughly mixed and incubated at the
appropriate temperature for 96 h in a chamber with temperature and humidity control.
Samples as whole flasks in triplicate were withdrawn after 96 h of fermentation (Francis et
al., 2002).
Optimization of Process Parameters in SSF
In a first step, the bagasse was milled to different particle sizes (<1.0, 1.0-2.0, 2.0-5.0,
5.0-10.0 and 10.0-15.0 mm) and used as support materials to determine the appropriate
particle size for maximal enzyme production in SSF. The initial moisture content of the
growth media was adjusted to 80% (Milagres et al., 2004) with a mixture of 2.5 mL of the
basal salt solution (initial pH of 6.0) and 16.25 mL of distilled water previously supplemented
with soluble potato starch to obtain the desire level of starch in the solid support (26 mg of
TS/g of dry sugar cane bagasse). The media were autoclaved (121ºC for 15 min), cooled and
inoculated with 1.25 mL of the spores suspension (1 × 10 7 spores/g of dry sugar cane
bagasse) previously prepared and then incubated at 30ºC for 96 h.
Secondly, a second-order orthogonal design (Box et al., 1989) was performed to asses the
effect of pH of the liquid medium (salt solution plus distilled water) and incubation
temperature on amylase production. The design consisted of 13 experiments with four (2 2 )
factorial points, four axial points to form a central composite design with α = 1.267 and five
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